Amino acids (AA) are the building block of protein. The basic function of dietary protein is to supply adequate amounts of required AA for the animal diets. The protein requirement for poultry is basically the requirement of AA, because AA are the end product of protein hydrolysis1. Proper body growth and tissue development of poultry need both essential and non-essential AA through the dietary supplement. As it is reported that indispensable AA can not be synthesized by the birds to meet up their protein requirement. For this reason, the diet is always fortified with synthetic AA to attain optimum performance of birds for meat and egg production.
Amongst the essential AA, methionine (Met) is very important and is commercially available as synthetic form for the diet formulation of poultry. Plant protein sources are very insignificant source of Met, which is regarded as the first limiting amino acid for the avian species2,3. In addition, the evaluation of protein sources is rendered based upon the amounts of the availability of three limiting amino acids (lysine, methionine and tryptophan)4. In this regard, the methionine warrants the higher amount than others for the optimum productivity of the broiler chicken.
Methionine and cystine are the sulphur containing AA. It is observed that marginal concentrations of total sulfur AA (Met and Cys) in animal diets could lead to a great demand and production of supplemental Met compounds and it takes 40% of total feed grade AA used for animal production2. Either DL-Met (99% purity powders) or an aqueous solution of 2-hydroxy-4-(methylthio) butanoic acid is used as the most conventional sources of supplemental Met in animal feeds5. For the synthesis of protein, Met as a main limiting AA can act as methyl donor, anti-oxidant and the precursor of several bioactive compounds say glutathione and taurine6-8. Further, it could play an important role for the development and health status of animals9,10. Met tends to have a greater rate of first pass metabolism in the gut than some other essential AA9. The significant splanchnic Met metabolism indicates that the gastrointestinal tract could have a functional requirement for Met11.
However, in poultry feed, DL-Met has long been used as a source of supplemental Met. Although D-Met is generally assumed as efficacious as L-Met for the growth of poultry due to the conversion to L-Met in the liver and kidneys12,13. D-Met is not utilized directly by the cells of the gastrointestinal tract until it is converted to L-Met in either the liver or kidneys. In fact, L-Met is the biologically functional form of Met readily absorbed by the intestinal cells and therefore, L-Met can directly deliver beneficial effects to the gastrointestinal tract of chickens compared with D-Met14.
The introduction of L-Met in poultry diets is not much conventional as like as DL-Met available in the market. There is a plethora of research on dietary DL-Met AA supplementation in broiler chickens. Much data are not available regarding supplementation of L-Met in poultry. Recently, feed-grade supplemental L-Met became available from a fermentation process, which provides opportunities to use a naturally occurring form of Met in animal feed. Further, L-Met could be used as an immediate source of Met, which can play an important role for intestinal or splanchnic metabolism. Therefore, we could speculate that inclusion of L-Met in broiler diet could have a greater potential for the growth performance, redox status, gastro-intestinal development and glutathione level of broiler in compared to use of DL-Met15.
Feed grade L-Met is a new commercial product in the feed industry. Research focusing on L-Met in broiler chicken, could play a significant role to boost up the poultry industry of the country. For this why, the present study was undertaken so that the findings retrieved from this study, would be a guideline to determine the recommended level of L-Met for broiler feed formulation. Feed grade L-Met supplement in conventional diet could have a potential to improve the productivity of the broiler production. Considering the above, the current study was attempted to investigate the impact of various levels of L-Met (feed grade) on the growth response, livability, gastro-intestinal development and gut morphology of broiler chicken.
MATERIALS AND METHODS
Ethical approval: The study was conducted following the guidelines of the research policy of Chattogram Veterinary and Animal Sciences University (CVASU) and approved by the Animal Ethics Committee of CVASU, Bangladesh [Approval no. CVASU/Dir(R&E). EC/2019/94(4)].
Animal husbandry and experimental design: A total of 216 (Cobb 500) day-old broiler chicks was procured from the local renowned hatchery weighing on an average of 45.70±0.38 g each. The chicks were weighed on receipt and then randomly assigned into four dietary treatment groups i.e. D0 (DL-Met), D1 (0.20% L-Met), D2 (0.25% L-Met) and D3 (0.30% L-Met) (D0, D1, D2 and D3), where each treatment was replicated six times with nine birds per replicate in a completely randomized design (CRD). Birds were raised in battery cages for the entire trial period (day 1-33). Cages were divided into 16 pens of equal size furnished with one feeder and one drinker. All the birds had a free access to the diet, along with ad libitum fresh, clean drinking water during the entire trial period. The birds were exposed to a continuous lighting program. All the birds were vaccinated against Ranikhet (New Castle Disease) and Gumboro disease, as per the schedule mentioned in Table 3, recommended by Department of Livestock Services (DLS), Chattogram, Bangladesh from where the vaccines (lyophilized) were collected.
Diet: Ready-made broiler starter (crumble) diet was procured from the local market and used to feed the birds up to 2 weeks (day 1-14) as an adjustment period. The proximate composition and reporting values of chemical composition of ready-made starter diet are shown in Table 1. After that, finisher or test diets (mash) were prepared manually and provided the birds for the remaining trial period i.e., from day 15-33 day. Four different test diets (D0, D1, D2 and D3) were formulated as per the requirements of NRC16, shown in Table 2. All the diets were iso-caloric and iso-nitrogenous. Control diet (D0) was formulated with the all feedstuffs without L-Met, whereas D1, D2 and D3 test diets were prepared with the supplementation of L-Met at the rate of 0.20, 0.25 and 0.30%, respectively. The composition and nutritive values (calculated and analyzed in the lab) of the formulated or test diets (finisher) are shown in the Table 2.
Data and sample collection: Mortality of bird was recorded as it occurred, while body weight and feed intake were recorded weekly for the calculation of body weight gain and feed conversion ratio (FCR). Livability was calculated from mortality of birds per replicate cage. Two birds per pen were selected randomly, weighed and killed humanely on day 33 to record the relative weights of gastro-intestinal organs (liver, pancreas, bursa of fabricius, heart, small intestine, proventriculus, gizzard, spleen) for assessing the gastro-intestinal development of the birds. Tissue samples (2 or 3cm) were also collected from the ileum, by killing two birds from each replicate cage, for the measurement of intestinal morphology such as villi height, villi width, crypt depth and surface area on day 33. Feed samples were also collected prior supplying to the birds to determine the nutritive value of the feeds.
Sample processing and analyses
Feed sample: Collected feed samples were processed by grinding with the help of coffee grinder machine thoroughly to analyze for dry matter (DM%), moisture%, crude protein (CP%), crude fiber (CF%), ether extract (EE%) and ash using standard laboratory procedure17. Dry matter was determined by oven dry method. Crude protein was recovered by Kjeldahl process. Ether Extract was quantified by Soxhlet apparatus. Ash was measured by igniting the pre-ashing sample on a Muffle furnace at a temperature of 600°C for four to 6 h. Additionally, calcium (Ca%) and phosphorus (P%) were determined by atomic absorption and spectrophotometry, respectively. Metabolizable energy (ME) was determined indirectly on the basis of true metabolizable energy (TME) contents of the feed samples, assuming that TME was 8% higher than the ME, as it is reported that TME is 5-10% higher than ME18.
Tissue samples processing for morphometric measurement of broiler chicken: Collected tissue segments were flushed with 0.9% saline solution to remove the contents and put in the 10% neutral-buffered formalin. After that, the samples were dehydrated, cleaned and paraffin embedded. Duplicates tissue segments were processed by the paraffin sectioning at a 4 μm thickness, placed on a glass slides. The slides were stained with hematoxylin-eosin and covered with cover-slip. Histological indices were analyzed under a computer-aided light microscopic using image analyzer. Measurements of villus height and crypt depth were taken only from sections, where the plane of section ran vertically from the top of villus to the base of an adjacent crypt. The villi height (from the crypt mouth to the villi tip), crypt depth (from the base up to the region of transition between the crypt and villi), villus width (½ of the villus length) were measured. The villi surface area (the area of the villous per length unit per bird) and villi height:crypt depth ratio were also calculated.
Statistical analyses: All collected data were subjected to analyzing by one way Analysis of Variance (ANOVA) using Minitab software (Minitab, Minitab Version, 16, 2000). The significance of differences between means was tested using the Duncan’s multiple range tests (DMRT). Statistical significance was considered at p≤0.05.
Growth responses of broiler chicken: The results showed that the feed intake (FI) and live weight (LW) of broiler chickens up to 21 days were not influenced (p = 0.87; p = 0.57) by dietary treatments except for 33 days (Table 4). From 1-33 days, the birds of D3 diet group consumed the highest feed (p = 0.01) compared to other groups. Live weight was also significantly (p = 0.038) greater for the broilers fed D3 diet than the birds fed other diets up to 33 day. The feed conversion ratio (FCR) of broilers was unaffected (p = 0.133) by dietary treatment from day 1-33 (Table 4). Birds on D3 diet group had a comparatively lower FCR value (1.56) than the other diet group, although the FCR value between treatment was statistically identical or not affected (p>0.05) throughout the trial period.